The goal of this research effort is to understand how various types of white blood cells recognize and respond to the presence of a microorganism or cancer cell in the body, or inappropriately recognize a normal component of the body (an auto-antigen). Our experiments are designed to provide a detailed understanding of how the substances (antigens) making up these microorganisms, cancer cells, or normal self-components, are made visible to the defending cells of the innate (anti-unspecific) or adaptive (antigen-specific) limbs of the host defense system cells and how recognition of infections or malignant cells is linked through complex cell-cell interactions to the induction of protective or self-destruction effector responses. Our previous work has described the events within a cell that bring together the antigen and MHC molecule and the cellular distribution of antigenic complexes within the body (antigen processing and presentation). We are now conducting studies primarily at the cell and tissue level to relate the physiology of antigen recognition to the development of effector function, immune memory, or tolerance and to add to this picture the activities of other cell types such as non-hematopoietic cells like fibroblastic reticular cells (FRCs). During the past year we have used these novel imaging methods in concert with classical cellular immune assays to discern the molecular mechanism by which the small adapter protein SAP controls sustained interaction of antigen-specific T and B-cells, as we reported in the past. Our studies revealed that SAP is key to the use in lymphoid-lymphoid cell and not lymphoid-myeloid cell interactions of SLAM-family adhesive interactions involving CD84 and Ly108. Our findings revealed that in contrast to T-dendritic cell interactions, T-B cell interactions were less integrin and more SLAM dependent after 10-15 minutes of interaction, explaining why we found that SAP-deficient T cells fail to adhere to B cells long enough to deliver help function. These findings have provided a new level of insight into molecular control of T cell-dependent humoral immune responses of the type that are critical for effective vaccine responses. In a second major effort, we extended our study combining intravital imaging, in vitro migration analysis, and flow cytometry, along with micro-CT and related tools for assessing bone structure, to explore the role of the lipid-signaling pathway involving S1P and its receptors S1P1 and S1P2 in control of the process of osteoclastogenesis. Osteoclasts are large multinucleate myeloid cells responsible for bone resorbtion. They form by fusion of bone surface-adherent osteoclast precursors that are cells in the monocytoid series. We previously showed that S1P and S1P1 played a role in regulating the efficiency with which osteoclast precursor myeloid cells remained attached to the bone surface for long enough to participate in osteoclast formation, with S1P1 signaling promoting the return of the precursors to the blood before maturation occurs. We now have evidence that S1P2 acts in the opposite fashion, promoting chemorepulsion and augmenting the egress of the precursors from the blood and movement towards the bone surface. However, S1P2 operates in a very different range of S1P concentrations from S1P1, allowing these two receptors and the gradient of S1P that exists between bone surface and blood to finely regulate the rate of movement of osteoclast precursors to and from the bone surface, where other signals involving chemokines and integrins contribute to the rate of osteoclast formation. This study thus provides novel insight into a pathway with a major role in the balance of bone devotion and destruction and points to a new target for interference with pro-osteoporotic processes. We are using our 2photon intravital imaging technology on a range of other projects investigating the intersection of the immune system with vaccines, infectious antigen sources, and dying cells in tissues. We are extending our earlier studies on the chemokine control of CD4-CD8 T cell interactions to better understand how these cell types collaborate in lymphoid tissue to generate optimal primary and memory cell-mediated immune responses. Our recent work suggests that different dendritic ells present antigen initially to the majority of CD4 vs. CD8 T cells, raising questions about where and when during the response the two cell types interact with the same antigen presenting cell. We have also acquired data on the role of Tregs in controlling effector vs. central memory cell formation, the site(s) of delivery of TLR-conjugate vaccines to diverse dendritic cell populations in straining lymph nodes, and on the differential location of nave vs. memory cells in lymphoid tissues. We have also invested in expanding our imaging technologies by developing methods that permit performing 7 or 8 color fluorescent immunohistochemistry on lymphoid and other tissues, collecting tiles images across entire tissue sections, and computationally analyzing the data to assign multiple stains to specific ells in a multidimensional solid-phase analog of flow cytometric phenotyping. This method is showing us the complex and heterogeneous, non-random distribution of dendritic cell subsets in lymphoid tissues and the different in how these cells capture antigen and present it to different T cell subsets. This methodology has potential applicability to human and NHP tissues. We are also examining the role of various innate receptors in signaling to the cells we are imaging, attempting to integrate the actions of microbial stimuli in our assessment of immune cell behavior in situ. This work includes a broad analysis of NLR-family receptor function, along with use of traditional TLR ligands as adjuvants.